1. Volume 57, Issue 5, Pages 812-823 (March 2015)
Error-Free DNA Damage Tolerance and Sister Chromatid Proximity during DNA Replication Rely on the Polα/Primase/Ctf4 Complex 
Marco Fumasoni, Katharina Zwicky, Fabio Vanoli, Massimo Lopes, Dana Branzei 
Molecular Cell 
Volume 57, Issue 5, Pages (March 2015)
DOI: /j.molcel
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2. Molecular Cell 2015 57, 812-823DOI: (10.1016/j.molcel.2014.12.038)
Copyright © 2015 The Authors Terms and Conditions

3. Figure 1 Ctf4 Facilitates Error-Free DDT
(A) sgs1 (HY1461) and sgs1 ctf4Δ (HY1472) cells were synchronized in G1 with alpha-factor (α) at 25°C prior to release at 30°C in media containing 0.033% MMS. Genomic DNA, extracted from samples collected at the indicated time points, was digested with NcoI and analyzed by 2D gel with a probe for ARS305. Schematic representation of major 2D gel signals, FACS, and X molecule quantification plots are displayed. The columns in the quantification graphs denote the data mean of two independent experiments and the bars indicate ranges.
(B) Spontaneous mutations rates at CAN1 locus (×10−7) in WT (FY0001), rev3Δ (HY0008), ctf4Δ (HY3466), and ctf4Δ rev3Δ (HY3468). Mutation rates and 95% confidence intervals were estimated using the maximum-likelihood method. Non-overlapping confidence intervals indicate statistical significance.
(C) WT (FY1000) and ctf4Δ (HY2193) cells were grown at 25°C and then shifted to 30°C for 2 hr in YPD or YPD containing 0.02% MMS. PCNA modifications were detected using a monoclonal antibody against PCNA. Ponceau staining serves as loading control. See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
Primase and Cohesin Mutants Are Characterized by Defects in Cohesion and Template Switching
(A) sgs1Δ (FY1060) and sgs1Δ scc1-73 (HY1934) cells were synchronized in G1 with alpha-factor (α) at 25°C and released in media containing 0.033% MMS at 37°C. The genomic DNA was digested with EcoRV-HindIII and analyzed by 2D gel with a probe against ARS305. FACS and X-molecule quantification plots are shown.
(B) sgs1 (HY1461) and sgs1 pri1-M4 (HY1457) were synchronized in G2/M with nocodazole (N) prior to release at 28°C in media containing 0.033% MMS. The genomic DNA was digested with EcoRV-HindIII and analyzed with a probe against ARS305. FACS and X-molecule quantification plots are displayed. In both (A) and (B), the columns in the quantification graphs denote the data mean of two independent experiments and the bars indicate ranges.
(C) Cohesion assay: WT (HY1788), ctf4Δ (HY1853), pri1-M4 (HY1872), chl1Δ (HY1823), and ctf18Δ (HY1825) cells were arrested in G1 at 25°C and released in nocodazole-containing medium for 3 hr at 30°C. The histogram shows the mean and SD of the percentage of cells showing two dots. The p value and asterisks indicating highly significant statistical difference are demonstrated for the pri1-M4 allele. The cohesion defects of chl1Δ, ctf4Δ, and ctf18Δ strains were also statistically significant, as previously reported (Xu et al., 2007). The differences in the cohesion defects of pri1-M4 and the chl1Δ, ctf4Δ, and ctf18Δ control strains were not statistically significant. Representation of the cells proficient (one dot; single) and deficient (two dots; double) in sister chromatid cohesion is shown. See also Figure S2.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3
Artificial Tethering of Sister Chromatids Suppresses the Template Switching Defects of Cohesin Mutants
(A) Schematic representation of the dimeric and tetrameric LacI systems for sister chromatid tethering.
(B) sgs1Δ and sgs1Δ scc1-73 cells carrying dimeric LacI (HY4252 and HY4255) or tetrameric LacI (HY4259 and HY4262) were grown at 25°C and synchronized in G2 by nocodazole (N) treatment. Cells were transferred to synthetic complete media lacking histidine and supplemented with 10 mM 3-aminotriazole and nocodazole for the last 20 min of the arrest to induce LacI expression. Cells were then released at 37°C in media containing 0.033% MMS, and samples were taken at the indicated time points. The genomic DNA was digested with EcoRV/XhoI and analyzed with a probe flanking the LEU2 locus. FACS and X molecule quantification plots are displayed. The columns in the quantification graphs denote the data mean of two independent experiments, and the bars indicate ranges. See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4
Artificial Cohesion Does Not Restore Efficient Template Switching in ctf4Δ Mutants
(A) sgs1 and sgs1 ctf4Δ cells carrying dimeric LacI (HY3979 and HY3981) or tetrameric LacI (HY3983 and HY3985) were grown and arrested as in Figure 3B. Cells were then released at 30°C in media containing 0.033% MMS. 2D gel analysis was conducted as in the Figure 3B. FACS and X molecule quantification plots are displayed. The columns in the quantification graphs denote the data mean of two independent experiments and the bars indicate ranges.
(B) Premature sister chromatid separation in sgs1 ctf4Δ cells carrying the dimeric and tetrameric version of the LacI. The experiment was conducted as in Figure 2C. See also Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
Recombination Pathways Supporting the Viability of CTF4 Mutants
(A–C) Tetrad dissection of ctf4Δ X sgs1 rad52Δ and ctf4Δ X sgs1 rad51Δ (A), ctf4Δ X rad59Δ (B), and ctf4Δ X rfa1-T11 (C) crossings. The expected genotypes are indicated. In (B), the line indicates elimination of superfluous lanes from the tetrad dissection plate image.
(D) Deletion rate assay: single colonies of WT, ctf4Δ, pri1-M4, rad51Δ, ctf4Δ rad51Δ, and pri1-M4 rad51Δ cells (obtained from crossing FY1162 carrying the direct repeat construct, with ctf4Δ rad51Δ and pri1-M4 rad51Δ, respectively) were suspended in water and diluted before being plated on YPD, 5-FOA and low-adenine plates. Red sectors/colonies characteristic of deletion events are shown. Intra-chromosomal deletion rates and 95% confidence intervals were estimated using the maximum-likelihood method. See also Figure S5.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6
Structural Mapping of the Replication Defects in ctf4Δ and pri1-M4
(A–C) Electron microscopy analysis of the replication intermediates: (A) ssDNA stretches at the forks, (B) internal ssDNA gaps, and (C) reversed forks. WT (FY1000), ctf4Δ (HY2193), and pri1-M4 (HY1607) cells were synchronized in nocodazole at 25°C prior to release at 30°C in MMS-containing medium (0.033%). Genomic DNA from cells collected at 90 min was psoralen crosslinked in vivo and enriched in RIs. Representative molecules, together with schematic representations, are shown. Quantifications of the indicated parameters are also displayed. Statistical analysis is based on ∼80 replication forks for each strain. The analysis was conducted twice on genomic DNA derived from independent experiments with reproducible results.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7
Hypothetical Model of DDT Orchestration in WT Cells versus Primase/Ctf4 Complex Mutants
Response of replication forks encountering DNA damage; parental DNA is shown in black, newly synthesized chromatids in blue, the DNA lesion is represented by a white star, and the RNA-DNA primer synthesized by Polα/Primase in orange. In WT cells, physiological repriming activity allows restart of replication downstream the lesion and then post-replicative tolerance of the damage by template switching. In Ctf4 and Primase mutants, defects in synthesizing a new primer generate uncoupling of the leading and lagging strands with the formation of longer ssDNA stretches at the fork. The exposure of ssDNA promotes the annealing of homologous sequences that may result in spontaneous deletion events and fork reversal. Reversed forks can be further processed or mediate replication fork restart by BIR or other annealing-mediated events. In addition, the Polα/Primase/Ctf4-defective complex may perform unscheduled repriming attempts that would result in an increase of internal ssDNA gaps that can be filled-in partially by template switching or TLS events.
Molecular Cell  , DOI: ( /j.molcel )
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